Detection of CLCF1 protein expression by flow cytometry

Cardiotrophin-like cytokine factor 1 (CLCF1) is an IL-6 family cytokine with neurotrophic and immuno-modulating functions. CLCF1 mRNA has been detected in primary and secondary lymphoid organs, and up-regulation of CLCF1 mRNA levels has been associated with the T helper (Th) 17 polarization. However, information regarding CLCF1 expression by immune cells at the protein level remains scarce. We have developed a methodology that uses a monoclonal antibody (mAb) directed against CLCF1 for the detection of human and mouse CLCF1 by flow cytometry. We have successfully detected CLCF1 protein expression in cells from the mouse pro-B cell line Ba/F3 that were transduced with CLCF1 cDNA. Interestingly, we found that the anti-CLCF1 mAb inhibits CLCF1 biological activity in vitro by binding to an epitope that encompasses site III of the cytokine. Moreover, we have detected CLCF1 expression in mouse splenic T cells, as well as in vitro differentiated Th1 cells. The specificity of the fluorescence signal was demonstrated using Clcf1-deficient lymphocytes generated using a conditional knock-out mouse model. The detection of CLCF1 protein by flow cytometry will be a valuable tool to study CLCF1 expression during normal and pathological immune responses.

In order to identify a suitable antibody to detect CLCF1 protein expression by flow cytometry, we first generated a derivative of the mouse pro-B cell line Ba/F3 expressing a protein C-tagged human CLCF1 construct (Ba/F3 hCLCF1 ProtC ).CLCF1 expression was confirmed by Western Blot (WB) using an anti-protein C tag mAb and an anti-CLCF1 mAb (Clone #138815) (Fig. 1a).After confirming CLCF1 expression by WB, we tested whether the anti-CLCF1 mAb could also be used to detect CLCF1 by flow cytometry on fixed and permeabilized Ba/F3 hCLCF1 ProtC cells.To do so, we assessed both indirect (i.e. using unlabelled anti-CLCF1 mAb with a fluorochrome-conjugated anti-mouse IgG; Fig. 1b) and direct detection with CF405M-conjugated (Fig. 1c) or Alexa Fluor 647-conjugated anti-CLCF1 mAb (Fig. 1d).Our results showed that CLCF1 is quantifiable by flow cytometry in permeabilized Ba/F3 hCLCF1 ProtC cells using both indirect and direct detection.We also demonstrated that the conjugated anti-CLCF1 mAb is compatible with two commercial fixation/permeabilization kits (eBioscience FoxP3/Transcription factor staining buffer set and BD Cytofix/Cytoperm fixation/permeabilization kit), as well as formaldehyde fixation/methanol permeabilization (Fig. 1e).

The anti-CLCF1 mAb can cross-react with mouse CLCF1
Human and mouse CLCF1 amino acid sequences are highly conserved with 97% identity 7 .We therefore investigated whether the anti-CLCF1 mAb, which is directed against human CLCF1, could cross-react with mouse CLCF1.While the fluorescence signal observed was slightly lower for mouse CLCF1, we showed that both human and mouse CLCF1 could be detected by flow cytometry in transduced Ba/F3 cells (Fig. 1f).This result indicates that the anti-CLCF1 mAb can cross-react with mouse CLCF1, which makes it a useful tool for the study of CLCF1 expression and function in both human and mouse tissues.

The anti-CLCF1 mAb interferes with the CLCF1-CRLF1 interaction
In order to be secreted, CLCF1 needs to form a composite cytokine complex with CRLF1 4 .We investigated whether the presence of CRLF1 could interfere with the detection of CLCF1 by flow cytometry.For this purpose, Ba/F3 cells were transduced with a CLCF1 cDNA or a bi-cistronic cDNA coding for both CRLF1 and CLCF1 (CRLF1-T2A-CLCF1).CRLF1 and CLCF1 expression was confirmed in transduced Ba/F3 cells by Taqman realtime quantitative PCR (RT-qPCR) (data not shown).Interestingly, despite blocking CRLF1-induced secretion of CLCF1 with brefeldin A, CLCF1 was undetectable in cells co-expressing CLCF1 and CRLF1 (Fig. 2a).This suggests that CRLF1 masks the epitope recognized by the anti-CLCF1 mAb, thus preventing the detection of CLCF1 in the CLCF1/CRLF1 complex (Fig. 2a).
Like IL-6 and CNTF, CLCF1 has three receptor binding sites 17 .To investigate the interaction interface between CLCF1 and the anti-CLCF1 mAb, we produced recombinant CLCF1 with site-specific mutations that inactivate CLCF1 binding site I (W67A) or site III (F151A/K154A) 17 .Previous studies have indicated that CLCF1 site I binds CNTFRα, while site III binds LIFRβ 17 .Mutation of CLCF1 binding site III also prevents CRLF1-mediated CLCF1 secretion 17 .We observed by WB that, while the mutation of CLCF1 binding site I has no effect, the anti-CLCF1 mAb is unable to detect the CLCF1 site III mutant (Fig. 2b).This data suggests that the epitope recognized by the anti-CLCF1 mAb encompasses the site III of CLCF1.
To further test whether the anti-CLCF1 mAb and CRLF1 both bind CLCF1 site III, we investigated the effect of the anti-CLCF1 mAb on the interaction between CLCF1 and CRLF1.To do so, we used Ba/F3 cells that were transduced with a retrovirus coding for a hCRLF1-sggg linker-GPI anchor chimeric fusion protein (i.e. a membrane-bound derivative of CRLF1).Using these cells, the binding of biotinylated CLCF1 to membranebound CRLF1 can be detected by flow cytometry using PE-conjugated streptavidin.Increasing concentrations of anti-CLCF1 mAb were added to determine whether the anti-CLCF1 mAb could inhibit the binding of CLCF1 to CRLF1.Our results showed a dose-dependent blocking of CLCF1 binding to CRLF1 in the presence of the CLCF1 can be detected by flow cytometry in Ba/F3 cells transduced with human and mouse CLCF1 cDNA.Ba/F3 cells were transduced with empty pMX retroviruses or pMX coding for protein C epitope-tagged human or mouse CLCF1 cDNA.(a) hCLCF1 expression was confirmed by Western Blot using mAbs specific for the tag (anti-ProtC) or for CLCF1 (anti-CLCF1).Ba/F3 cells transduced with empty pMX retroviruses (Ba/ F3 empty vector) and recombinant hCLCF1 produced in E. coli (10 ng) were used as controls.The molecular weight difference between the recombinant hCLCF1 and the hCLCF1 from Ba/F3 cells is seemingly due to posttranscriptional modifications in eukaryote cells.Western Blot images were cropped.Original blots are presented in Fig. S4.(n = 5) (b-d) Cells were fixed and permeabilized with formaldehyde and methanol.Flow cytometry fluorescence signal in control (filled grey histograms) or hCLCF1 (black line histograms) expressing Ba/F3 cells was obtained using (b) indirect detection with an anti-CLCF1 mAb followed by an Alexa Fluor 488-conjugated secondary antibody (n = 4) or (c,d) direct detection with an anti-CLCF1 mAb conjugated to (c) CF405M (n = 7) or (d) Alexa Fluor 647 (n = 5).Control stainings with secondary antibodies and/or unstained cells are presented in Fig. S3a-c.(e) Cells were fixed and permeabilized with formaldehyde and methanol (left panel), the eBioscience FoxP3/Transcription factor staining buffer set (middle panel) or the BD Cytofix/Cytoperm fixation/ permeabilization kit (right panel).Flow cytometry fluorescence signal was detected in control (filled grey histogram) or hCLCF1 expressing (black line histogram) Ba/F3 cells stained with an Alexa Fluor 647-conjugated anti-CLCF1 mAb.(n = 3) (f) Cells were fixed and permeabilized with formaldehyde and methanol.Flow cytometry fluorescence signal was detected in control (filled grey histogram), human (dotted line histogram) or mouse (solid line histogram) CLCF1 expressing Ba/F3 cells that were stained with a CF405M-conjugated anti-CLCF1 mAb.(n = 3).www.nature.com/scientificreports/anti-CLCF1 mAb.Indeed, we observed a partial block in the presence of 1 μg/ml of anti-CLCF1 mAb (0.67 molar ratio between anti-CLCF1 mAb and CLCF1) or 5 μg/ml (3.3 molar ratio), and a near complete block in the presence of 10 μg/ml (6.7 molar ratio) (Fig. 2c).This data shows that the anti-CLCF1 mAb can prevent CLCF1-CRLF1 interaction, likely by competing with CRLF1 for binding on CLCF1 site III.www.nature.com/scientificreports/

The anti-CLCF1 mAb can inhibit CLCF1 biological activity in vitro
In addition to its role in the CLCF1-CRLF1 interaction, CLCF1 binding site III is also involved in the recruitment and activation of the LIFRβ receptor chain of the tripartite CNTFR 17 .Since the anti-CLCF1 mAb seems to block CLCF1 site III, we hypothesized that the anti-CLCF1 mAb could inhibit CLCF1 biological activity on its receptor by blocking the interaction with the receptor signaling chain LIFRβ.To test this, we used Ba/F3 cells that were transduced with cDNAs coding for the 3 subunits of the CNTFR (i.e.CNTFRα/LIFRβ/gp130).In these cells, activation of the CNTFR with cytokines leads to the activation of the JAK/STAT3 signaling pathway, which induces cell proliferation.Using these CNTFR-expressing Ba/F3 cells, we demonstrated that 2.5 μg/ml of anti-CLCF1 mAb (3.3 molar excess) is sufficient to produce a significant reduction in STAT3 phosphorylation (Fig. 3a), and that this decrease in CNTFR activation is associated with a matching reduction in CLCF1-induced proliferation (Fig. 3b).Altogether, these results show that the anti-CLCF1 mAb can inhibit CLCF1 activity in vitro.

Detection of CLCF1 in mouse T cells
We next sought to utilize our CLCF1 detection methodology to investigate CLCF1 expression in mouse splenocytes.To assess the specificity of the flow cytometry fluorescence signal, we used splenocytes from Clcf1 conditional knock-out mice.This mouse model was obtained by breeding Clcf1 flox/flox mice (i.e.mice that had LoxP sites inserted either side of the exon 3 in the Clcf1 gene using CRISPR-Cas9 technology 18 ) with Vav-iCre transgenic mice to generate offspring with a constitutive knock-out of Clcf1 in immune cells.Taqman RT-qPCR was used to confirm the Vav-iCre induced deletion of Clcf1 in our KO splenocytes (Fig. S1).
Using our flow cytometry CLCF1 detection protocol with the Clcf1 KO cells as control, we observed CLCF1 expression in total T cells (Fig. 4a upper panel), as well as in CD4 + and CD8 + T cells (Fig. 4a middle and lower panels).We also detected CLCF1 expression in B cells, myeloid cells, and NK cells (Fig. S2).To investigate whether CLCF1 expression in T cells is modulated by activation, we stimulated splenocytes with plate-bound anti-TCR and soluble anti-CD28 for 3 d.The expression pattern of CLCF1 was similar in naïve and activated cells, with expression in both CD4 + and CD8 + T cells (Fig. 4b).
As CRLF1 promotes CLCF1 secretion 4 and interferes with CLCF1 detection by the anti-CLCF1 mAb (Fig. 2c), we also assessed CRLF1 mRNA levels by RT-qPCR in unfractionated splenocytes, as well as resting and activated CD4 + T cells from wild-type mice.Our results show that CRLF1 expression was minimal/undetectable in all tested conditions with cycle threshold (Ct) values over 37 (data not shown).

Detection of CLCF1 in mouse Type 1 helper T cells
The expression levels of many cytokines are altered during Th cell differentiation.CLCF1 is no exception as CLCF1 mRNA is over-expressed in human memory Th17 cells expanded in vitro 14 .To investigate whether our methodology could detect CLCF1 expression in Th cell subtypes, we cultured mouse T cells under non-polarizing conditions (Th0) or conditions favoring Th1 polarization.To induce Th1 polarization, we stimulated CD4 + T cells from WT or Clcf1 conditional KO mice with anti-TCR and anti-CD28 in the presence of anti-IL-4 and IL-12 for 5 d.By comparing the fluorescence signal between the WT and KO cells, we were able to detect CLCF1 expression in cells differentiated under non-polarizing (Th0) and Th1 polarizing conditions (Fig. 5).These results indicate that CLCF1 can be detected in CD4 + effector T cells, and validates the use of anti-CLCF1 mAb as a tool to study CLCF1 expression patterns in T cell subpopulations.

Discussion
Our aim was to develop a mAb-based detection protocol for the cytokine CLCF1 that could easily be combined with other mAbs in multi-color panel flow cytometry.It represents a substitute for our previous approach, which used rabbit polyclonal anti-CLCF1 serum to show CLCF1 expression in a subpopulation of GR-1 + cells in the mouse bone marrow 16 .With this current methodology, we managed to detect CLCF1 expression in both unstimulated and activated mouse splenic CD4 + and CD8 + T cells, as well as in in vitro differentiated Th1 cells.Our approach was validated using transduced mouse Ba/F3 cells overexpressing CLCF1, and primary immune cells deficient for Clcf1.RT-qPCR results on splenocytes from Clcf1 KO mice indicated that CLCF1 mRNA is undetectable in these cells, thus confirming the effectiveness of the Clcf1 deletion in hematopoietic cells by the Vav-iCre promoter.As the complete KO of Clcf1 in mice is lethal at P1 19 , this new mouse model will be useful for the future study of CLCF1 immune functions and pathological roles.CLCF1 was first cloned from activated Jurkat human T cell lymphoma cells 2 .This is consistent with our current observations of CLCF1 expression in resting and activated mouse T cells.Our investigation further showed that this cytokine is expressed in Th1 cells.We believe our methodology could be used in the future to further characterize CLCF1 expression patterns at various stages of T cell activation, as well as in other types of T helper cell differentiation.
Conversely, CLCF1 secretion partner CRLF1 does not seem to be expressed by T cells, as CRLF1 mRNA levels were at the detection limit of RT-qPCR in both splenocytes and CD4 + T cells.Therefore, CLCF1 produced by T cells is unlikely to be secreted via the CLCF1-CRLF1 complex.It remains to be investigated whether CLCF1 in T cells could be secreted in complex with an alternative secretion partner 20 , or released via an unconventional pathway as shown for IL-35 21 , which would allow this cytokine to mediate its known immuno-modulating activities on B cells 1,2 or myeloid cells 8 .Additionally, intracellular CLCF1 in T cells might possess yet to be identified intracrine roles in the control of Th cell differentiation.
Moreover, we showed using CLCF1 mutants that the epitope recognized by the anti-CLCF1 mAb overlaps with CLCF1 binding site III.This site is responsible for the interaction of CLCF1 with either CRLF1 for secretion or LIFRβ for receptor activation 17 .We showed that the anti-CLCF1 mAb cannot detect CLCF1 when its binding site III is masked by CRLF1 in the CLCF1/CRLF1 complex.As CLCF1 could be detected in T cells by flow cytometry, this observation further suggests that the cytokine is not complexed with CRLF1 in these cells.Furthermore, the anti-CLCF1 mAb can prevent the formation of the CLCF1/CRLF1 complex, as well as inhibit the activation of CNTFR, presumably by blocking the recruitment of LIFRβ.CLCF1 is currently being investigated as a potential target for the treatment of various types of cancers, such as gliomas 22 and non-small cell lung cancer 13 .The group of Dr. Sweet-Cordero is also developing an engineered high-affinity soluble form of CNTFRα (eCNTFR-Fc) that has been shown to sequester CLCF1, therefore inhibiting its oncogenic effects in a lung adenocarcinoma mouse model 23 .Our results suggest that the anti-CLCF1 mAb, which recognizes CLCF1 site III, could potentially be used as an alternative for the neutralization of CLCF1 oncogenic activity in these preclinical models.Moreover, some pathologies, such as focal segmental glomerulosclerosis 24 , might implicate CLCF1 rather than the CLCF1/CRLF1 complex.The anti-CLCF1 mAb inability to bind CLCF1 when in complex with CRLF1 therefore makes it a candidate for the preclinical investigation of selective inhibition of free CLCF1 in these pathologies.
In conclusion, the methodology outlined in this study allows the investigation of normal and pathological CLCF1 expression patterns at a protein level, which will facilitate the study of CLCF1 function in normal or pathological settings, and potentially reveal new roles for this understudied cytokine.

Experimental animals
The Clcf1 floxed C57BL/6 mouse model with LoxP sites inserted in 5' and 3' of the Clcf1 third exon 18 was crossbred to wild-type C57BL/6 mice (Strain #000664) for 6 generations.Clcf1 flox/flox mice were then bred to C57BL/6 Vav-iCre transgenic mice (Strain #008610) 25 purchased from The Jackson Laboratory (Bar Harbor, ME) to produce experimental animals that were used at 8-12 weeks of age.Age and sex-matched wild-type C57BL/6 mice were used as controls.All procedures were performed in accordance with the Canadian Council on Animal Care guidelines and approved by the Comité de déontologie de l' expérimentation sur les animaux (CDEA),

Detection of CLCF1 and CRLF1 mRNA
Mouse unfractionated splenocytes or CD4 + T cells were lysed in TRIzol (Thermo Fisher Scientific), chloroform was added, and total RNA was isolated from the aqueous phase using the RNeasy kit (Qiagen, Toronto ON) according to manufacturer's instructions.TaqMan real-time quantitative RT-PCR analysis was conducted by the Genomics platform at the Institute for Research in Immunology and Cancer (Université de Montréal, Montréal QC).

Statistical analysis
Unpaired Student's t tests or one-way ANOVAs with a Bonferroni or Tukey post-hoc test were used where appropriate.The results presented in the figures are marked using asterisks with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.Data were analyzed using the GraphPad prism software (La Jolla CA).

Figure 1 .
Figure1.CLCF1 can be detected by flow cytometry in Ba/F3 cells transduced with human and mouse CLCF1 cDNA.Ba/F3 cells were transduced with empty pMX retroviruses or pMX coding for protein C epitope-tagged human or mouse CLCF1 cDNA.(a) hCLCF1 expression was confirmed by Western Blot using mAbs specific for the tag (anti-ProtC) or for CLCF1 (anti-CLCF1).Ba/F3 cells transduced with empty pMX retroviruses (Ba/ F3 empty vector) and recombinant hCLCF1 produced in E. coli (10 ng) were used as controls.The molecular weight difference between the recombinant hCLCF1 and the hCLCF1 from Ba/F3 cells is seemingly due to posttranscriptional modifications in eukaryote cells.Western Blot images were cropped.Original blots are presented in Fig.S4.(n = 5) (b-d) Cells were fixed and permeabilized with formaldehyde and methanol.Flow cytometry fluorescence signal in control (filled grey histograms) or hCLCF1 (black line histograms) expressing Ba/F3 cells was obtained using (b) indirect detection with an anti-CLCF1 mAb followed by an Alexa Fluor 488-conjugated secondary antibody (n = 4) or (c,d) direct detection with an anti-CLCF1 mAb conjugated to (c) CF405M (n = 7) or (d) Alexa Fluor 647 (n = 5).Control stainings with secondary antibodies and/or unstained cells are presented in Fig.S3a-c.(e) Cells were fixed and permeabilized with formaldehyde and methanol (left panel), the eBioscience FoxP3/Transcription factor staining buffer set (middle panel) or the BD Cytofix/Cytoperm fixation/ permeabilization kit (right panel).Flow cytometry fluorescence signal was detected in control (filled grey histogram) or hCLCF1 expressing (black line histogram) Ba/F3 cells stained with an Alexa Fluor 647-conjugated anti-CLCF1 mAb.(n = 3) (f) Cells were fixed and permeabilized with formaldehyde and methanol.Flow cytometry fluorescence signal was detected in control (filled grey histogram), human (dotted line histogram) or mouse (solid line histogram) CLCF1 expressing Ba/F3 cells that were stained with a CF405M-conjugated anti-CLCF1 mAb.(n = 3). https://doi.org/10.1038/s41598-024-64101-9

Figure 2 .
Figure 2. The anti-CLCF1 mAb recognizes an epitope that encompasses CLCF1 binding site III and can inhibit CLCF1-CRLF1 interaction.(a) Ba/F3 cells were transduced with empty pMX retroviruses (empty vector; filled grey histogram) or pMX coding for human CLCF1 (hCLCF1; dotted line histogram) or human CLCF1 and CRLF1 (hCRLF1-T2A-hCLCF1; solid line histogram).CRLF1-induced CLCF1 secretion was inhibited by adding brefeldin A for 4 h and cells were fixed and permeabilized with formaldehyde and methanol.Flow cytometry fluorescence signal was detected with a CF405M-conjugated anti-CLCF1 mAb.(n = 4) (b) Recombinant wild-type CLCF1 (hCLCF1), binding site I mutant CLCF1 (hCLCF1 W67A ) or binding site III mutant CLCF1 (hCLCF1 F151A/K154A ) were produced in E. coli and subjected to Western Blot analysis using anti-ProtC (upper panel) or anti-CLCF1 (lower panel) mAbs.Western Blot images were cropped.Original blots are presented in Fig. S4.(n = 3) (c) Ba/F3 cells were transduced with a pMX derivative coding for a hCRLF1-sggg linker-GPI anchor fusion protein and incubated with biotinylated hCLCF1 in the presence of anti-CLCF1 mAb (solid line histogram) or isotype control (dotted line histogram).Binding of CLCF1 to cell surface-bound CRLF1 was detected using PE-conjugated streptavidin.Fluorescence signal of PE-conjugated streptavidin background binding in the absence of hCLCF1 is used as a negative control (filled grey histogram).Bar graphs represent mean fluorescence intensity (MFI) of PE-conjugated streptavidin ± SEM.Statistical significance was assessed using an ANOVA with a Bonferroni post-hoc test, with **p < 0.01 and ****p < 0.0001.(n = 6).

Figure 3 .
Figure 3.The anti-CLCF1 mAb can inhibit CLCF1 biological activity in vitro.Ba/F3 cells were transduced with pMX retroviruses coding for the three receptor chains (CNTFRα, LIFRβ and gp130) of the CNTF receptor (Ba/ F3 CNTFR).(a) Ba/F3 CNTFR triplicate cell cultures were stimulated with hCLCF1 (25 ng/ml) in the presence of vehicle, isotype control or anti-CLCF1 mAb (2.5μg/ml).Cell lysates were subjected to Western Blot analysis with mAbs specific for phosphorylated (upper panel) or total STAT3 (lower panel).Western Blot signal intensity was quantified using the ImageJ software.Western Blot images were cropped.Original blots are presented in Fig. S4.Bar graph represents pSTAT3 signal intensity ± SEM.Statistical significance was assessed using an ANOVA with a Bonferroni post-hoc test, with ***p < 0.001 and ****p < 0.0001.(n = 3) (b) Proliferation of Ba/F3 CNTFR cells in response to increasing concentrations of hCLCF1 was assessed in the presence of 2.5 μg/ml of anti-CLCF1 mAb (red triangles), isotype control (black squares) or vehicle (grey circles).Ba/F3 proliferation was measured in triplicates using an alamarBlue fluorometric assay.Statistical significance was assessed using a oneway ANOVA and a Tukey post-hoc test for each concentration point of hCLCF1, with *p < 0.05 and **p < 0.01.(n = 5).

Figure 4 .
Figure 4. CLCF1 is expressed by mouse CD4 + and CD8 + T cells.Mouse splenocytes were analysed (a) upon isolation or (b) after 3 d of activation with plate-bound anti-TCR and soluble anti-CD28.(a,b) All cells were stimulated with PMA/ionomycin/brefeldin A for 4 h, and fixed and permeabilized with the eBioscience FoxP3/ Transcription factor staining buffer set.The panels on the left show the immune population investigated (top : CD3 + ; middle and bottom : CD4 + and CD8 + gated on CD3 + cells).The panels to the right compare the fluorescence signal of the Alexa fluor 647-conjugated anti-CLCF1 mAb of cells isolated from Clcf1 knock-out mice (KO; filled grey histogram) or wild-type mice (WT; black line histogram).The bar graphs show the relative fluorescence intensity (RFI) for the Alexa Fluor 647-conjugated anti-CLCF1 mAb ± SEM.Statistical significance was assessed using Student's t test, with *p < 0.05, **p < 0.01, and ****p < 0,0001.Control unstained cells are presented in Fig. S3d,e.Experiments were performed in triplicate, and replicated in three separate experiments.

Figure 5 .
Figure 5. CLCF1 is expressed by differentiated Th1 cells.Mouse splenic CD4 + T cells were activated with plate-bound anti-TCR and soluble anti-CD28 alone (Th0) or supplemented with neutralizing anti-IL-4 mAb and mouse IL-12 (10 ng/ml) (Th1) for 5 d.Cells were re-stimulated with PMA/ionomycin/brefeldin A for 4 h, and fixed and permeabilized with the eBioscience FoxP3/Transcription factor staining buffer set.A flow cytometry histogram graph and a bar graph are shown for each stimulation condition (Left : Th0; Right : Th1).The flow cytometry histogram graph compares the fluorescence signal of the Alexa Fluor 647-conjugated anti-CLCF1 mAb of cells isolated from a Clcf1 KO (grey filled histogram) or WT (black line histogram) mouse.The bar graph represents relative fluorescence intensity (RFI) of the Alexa Fluor 647-conjugated anti-CLCF1 mAb ± SEM.Statistical significance was assessed using Student's t test, with **p < 0.01 and ****p < 0.0001.Control unstained cells are presented in Fig. S3f.The experiment was performed in triplicate, and replicated in three separate experiments.